The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Pecher, Pascal; Arnold, Ulrich

doi:10.1016/j.bpc.2008.12.005

Cited by 35 publications

(42 citation statements)

References 71 publications

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“…Destabilization of protein stability through the introduction of additional disulfide bridges has been reported in one or some mutants of Candida antarctica lipase B (10), Drosophila melanogaster acetylcholinesterase (26), and RNase A (27).…”

Section: Figmentioning

confidence: 99%

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu

Deng

Yang

et al. 2014

Appl Environ Microbiol

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High thermostability is required for alkaline ␣-amylases to maintain high catalytic activity under the harsh conditions used in textile production. In this study, we attempted to improve the thermostability of an alkaline ␣-amylase from Alkalimonas amylolytica through in silico rational design and systems engineering of disulfide bridges in the catalytic domain. Specifically, 7 residue pairs (P35-G426, Q107-G167, G116-Q120, A147-W160, G233-V265, A332-G370, and R436-M480) were chosen as engineering targets for disulfide bridge formation, and the respective residues were replaced with cysteines. Three single disulfide bridge mutants-P35C-G426C, G116C-Q120C, and R436C-M480C-of the 7 showed significantly enhanced thermostability. Combinational mutations were subsequently assessed, and the triple mutant P35C-G426C/G116C-Q120C/R436C-M480C showed a 6-fold increase in half-life at 60°C and a 5.2°C increase in melting temperature compared with the wild-type enzyme. Interestingly, other biochemical properties of this mutant also improved: the optimum temperature increased from 50°C to 55°C, the optimum pH shifted from 9.5 to 10.0, the stable pH range extended from 7.0 to 11.0 to 6.0 to 12.0, and the catalytic efficiency (k cat /K m ) increased from 1.8 ؋ 10 4 to 2.4 ؋ 10 4 liters/g · min. The possible mechanism responsible for these improvements was explored through comparative analysis of the model structures of wild-type and mutant enzymes. The disulfide bridge engineering strategy used in this work may be applied to improve the thermostability of other industrial enzymes.

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Section: Figmentioning

confidence: 99%

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Liu

Deng

Yang

et al. 2014

Appl Environ Microbiol

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“…Proteolysis of RNase A and numerous variants thereof had yielded substantial information on the stability and unfolding of this enzyme. 5,[18][19][20][21] Here, we exploited the high efficiency of thermolysin to degrade thermally Figure 1. Ribbon diagram of wild-type RNase A (PDB entry 7rsa 6 ) and structures of reverse turns relevant to this work.…”

Section: Thermally Induced Unfoldingmentioning

confidence: 99%

“…The signal was fitted by nonlinear regression as described previously. 20,34 The enthalpy and the entropy at T m (DH m and DS m , respectively) were determined with the van't Hoff equation.…”

Section: Thermally Induced Transitionmentioning

confidence: 99%

“…SDS-PAGE and densitometric evaluation of the RNase A bands were carried out as described previously. 18,20 The rate constants of proteolysis were determined from the decrease in the peak areas of the intact RNase A band, which followed a first-order reaction. Under the conditions applied, these values correspond to the respective unfolding rate constant, k U .…”

Section: Gdn-hcl-induced Transitionmentioning

confidence: 99%

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Protein prosthesis: β‐peptides as reverse‐turn surrogates

et al. 2013

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The introduction of non-natural modules could provide unprecedented control over folding/unfolding behavior, conformational stability, and biological function of proteins. Success requires the interrogation of candidate modules in natural contexts. Here, expressed protein ligation is used to replace a reverse turn in bovine pancreatic ribonuclease (RNase A) with a synthetic b-dipeptide: b 2 -homoalanine-b 3 -homoalanine. This segment is known to adopt an unnatural reverse-turn conformation that contains a 10-membered ring hydrogen bond, but one with a donor-acceptor pattern opposite to that in the 10-membered rings of natural reverse turns. The RNase A variant has intact enzymatic activity, but unfolds more quickly and has diminished conformational stability relative to native RNase A. These data indicate that hydrogen-bonding pattern merits careful consideration in the selection of beneficial reverse-turn surrogates.

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“…For instance, the failure of designed disulfide bridges to stabilize some proteins has been related to the irreversible, kinetically-controlled nature of the denaturation process [20]. However, this interpretation remains unproven, given the low level of success often met in the rational design of stabilizing disulfide bridges [21], [22].…”

Section: Introductionmentioning

confidence: 99%

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

et al. 2013

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The impact of disulfide bonds on protein stability goes beyond simple equilibrium thermodynamics effects associated with the conformational entropy of the unfolded state. Indeed, disulfide crosslinks may play a role in the prevention of dysfunctional association and strongly affect the rates of irreversible enzyme inactivation, highly relevant in biotechnological applications. While these kinetic-stability effects remain poorly understood, by analogy with proposed mechanisms for processes of protein aggregation and fibrillogenesis, we propose that they may be determined by the properties of sparsely-populated, partially-unfolded intermediates. Here we report the successful design, on the basis of high temperature molecular-dynamics simulations, of six thermodynamically and kinetically stabilized variants of phytase from Citrobacter braakii (a biotechnologically important enzyme) with one, two or three engineered disulfides. Activity measurements and 3D crystal structure determination demonstrate that the engineered crosslinks do not cause dramatic alterations in the native structure. The inactivation kinetics for all the variants displays a strongly non-Arrhenius temperature dependence, with the time-scale for the irreversible denaturation process reaching a minimum at a given temperature within the range of the denaturation transition. We show this striking feature to be a signature of a key role played by a partially unfolded, intermediate state/ensemble. Energetic and mutational analyses confirm that the intermediate is highly unfolded (akin to a proposed critical intermediate in the misfolding of the prion protein), a result that explains the observed kinetic stabilization. Our results provide a rationale for the kinetic-stability consequences of disulfide-crosslink engineering and an experimental methodology to arrive at energetic/structural descriptions of the sparsely populated and elusive intermediates that play key roles in irreversible protein denaturation.

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The effect of additional disulfide bonds on the stability and folding of ribonuclease A

Cited by 35 publications

References 71 publications

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

In Silico Rational Design and Systems Engineering of Disulfide Bridges in the Catalytic Domain of an Alkaline α-Amylase from Alkalimonas amylolytica To Improve Thermostability

Protein prosthesis: β‐peptides as reverse‐turn surrogates

Mechanism of Protein Kinetic Stabilization by Engineered Disulfide Crosslinks

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